Strategic loci on the hemoglobin molecule, such as the N-termini of the alpha and beta chains, will be chemically modified with derivatives of pyridoxal. In this way the oxygen affinity of hemoglobin will be modulated over a wide range. A specific reagent which crosslinks the beta chains at the polyphosphate binding site, i.e. 2-nor-2-formylpyridoxal 5'-phosphate, will serve as a powerful tool for investigating the role of subunit dissociation in such reactions as cooperative ligand binding and conformational change, the hemoglobin-haptoglobin reaction, and many others. The structural details of the crosslinked hemoglobin will be investigated by X-ray crystallography and NMR spectroscopy to reach an understanding of the special functional properties of this molecule. We will also continue our study of hemoglobin molecules composed of four identical subunits. The apparent asymmetry of these tetramers, suggested by the presence of only one polyphosphate binding site, will be explored with the aid of reagents specific for the alpha and beta N-termini of normal hemoglobin, as well as with the new specific reagent for crosslinking beta chains. Finally, we shall investigate the linkage between proton and organic phosphate binding to hemoglobin both theoretically and experimentally. In addition, we propose to study the properties of some new allosteric effectors in which sulfate esters replace the normal phosphate ones.